Alkyl nitrates are added to commercial diesel fuels in order to guarantee their cetane number (Suppes et al., Energy & Fuels 15, 151-157, 2001). The cetane number is an auto-ignition characteristic of diesel fuels. According to engine manufacturers, a cetane number of 50 is an acceptable minimum value for combustion to be controlled satisfactorily. In the case of direct-injection engines, the cetane number also conditions the noise delivered by the engine (Guibet J. C. et al., Carburants et Moteurs, Editions Technip, Paris (1999)).
2-EHN is the most commonly used procetane additive. Liquid at ambient temperature, this compound decomposes autocatalytically above 100° C., releasing free radicals that reduce the auto-ignition delay of the engine. 2-EHN is added to fuels in a proportion of 300 to 1000 mg/L, which explains why 105 tons of this compound are produced yearly worldwide. 2-EHN is the most commonly used procetane additive.
2-EHN is the nitric ester of a branched alcohol, 2-ethylhexanol. Its structure is as follows:

Its main physico-chemical properties are given in Table 1.
TABLE 1Main physico-chemical properties of 2-EHN*CharacteristicValueCAS number27247-96-7Molar mass175.2gDensity0.96kg/LVapour pressure at 20° C.27PaSolubility at 20° C.12.6mg/LLog KO/W5.24*According to “The American Chemistry Council Petroleum Additives Panel Health, Environmental and Regulatory Task Group”, October 2006
Diesel fuel is 88% biodegradable by the microflora of soils polluted by hydrocarbons (Penet et al., Appl. Microbiol. Biotechnol., 66, 40-47, 2004). However, the acceptability of 2-EHN is a topical issue, especially within the context of the European directive REACH (Chemical Consulting Global Laboratory Network). In fact, no data relative to the biodegradability of 2-EHN is available.
On the other hand, the ecotoxicity properties of 2-EHN are known. The acute toxicity of 2-EHN is low since the LC50 (50% lethal concentrations) for zebrafish and Daphnia are higher than its water solubility. However, 2-EHN is much more dangerous when administered in repeated doses. In rats, the level at which no unfavourable effect is observed after 28 days (No Observed Adverse Effect Level or NOAEL) is only 28 mg/kg·day (Someroja et al., Toxicol. Lett., 19, 189-93, 1983). This result shows that the biodegradability of 2-EHN is a key parameter as regards global risk in case of accidental release. In fact, biodegradability determines the critical time of exposure to the dangerous molecule.
Sturm's test (Sturm, Journal of Oil Chemistry Society, 50, 159-167, 1973) is the most commonly used normalized test for determining the biodegradation of a compound. This test, carried out in a liquid medium, is fast and easy to conduct. It is performed in a closed system where the compound to be tested is subjected to the cellular biomass consisting of a sewage disposal plant sludge. The degradation is then measured by means of the final production of CO2 trapped by a base. According to Battersby et al. (Chemosphere, 38, 3219-3235, 1999), Sturm's test is applicable only to the water-soluble organic products of low volatility. The biodegradability test corresponding to that of the CO2 headspace was used for 2-EHN. This test showed that 2-EHN is not readily biodegradable according to the usual criteria that impose 60% biodegradation within 28 days with, at least, 10% biodegradation within 10 days. According to the authors of a report from the American Chemistry Council (2006) concerning 2-EHN, the absence of biodegradability therefore has to be attributed to the volatility of the substrate. Considering the small amounts of substrate used in this normalized test, the initial 2-EHN used as substrate is mainly located in the headspace of the test jar and it is then not available for the microflora. This test is therefore not pertinent for 2-EHN.
It is known that substituted hydrocarbons often show reluctance to biodegradation. 2-EHN has a branched structure and it is consequently not or very poorly degraded by the sewage disposal plant microflora that are conventionally used in biodegradation tests.
We have discovered a bacterium allowing to degrade 2-EHN and adapted a test allowing to measure the biodegradability of this compound.